Synthesis of 3-((4-Hydroxyphenyl)amino)propanoic Acid Derivatives as Promising Scaffolds for the Development of Antimicrobial Candidates Targeting Multidrug-Resistant Bacterial and Fungal Pathogens

Infections caused by multidrug-resistant bacterial and fungal pathogens represent a significant global health concern, contributing to increased morbidity and mortality rates. Therefore, it is crucial to develop novel compounds targeting drug-resistant microbial strains. Herein, we report the synthesis of amino acid derivatives bearing an incorporated 4-hydroxyphenyl moiety with various substitutions. The resultant novel 3-((4-hydroxyphenyl)amino)propanoic acid derivatives 2–37 exhibited structure-dependent antimicrobial activity against both ESKAPE group bacteria and drug-resistant Candida species. Furthermore, these derivatives demonstrated substantial activity against Candida auris, with minimum inhibitory concentrations ranging from 0.5 to 64 µg/mL. Hydrazones 14–16, containing heterocyclic substituents, showed the most potent and broad-spectrum antimicrobial activity. This activity extended to methicillin-resistant Staphylococcus aureus (MRSA) with MIC values ranging from 1 to 8 µg/mL, vancomycin-resistant Enterococcus faecalis (0.5–2 µg/mL), Gram-negative pathogens (MIC 8–64 µg/mL), and drug-resistant Candida species (MIC 8–64 µg/mL), including Candida auris. Collectively, these findings underscore the potential utility of the novel 3-((4-hydroxyphenyl)amino)propanoic acid scaffold for further development as a foundational platform for novel antimicrobial agents targeting emerging and drug-resistant bacterial and fungal pathogens.


Introduction
Antimicrobial resistance remains one of the biggest public health threats worldwide.The emergence of multidrug-resistant bacterial pathogens remains a growing concern, resulting in an increase in morbidity and mortality [1,2].The World Health Organization (WHO) identified priority pathogens that are able to evade or escape conventional antimicrobial therapy due to their acquired resistance mechanisms and high virulence or pathogenicity [3][4][5].These pathogens include Enterococcus faecium, Staphylococcus aureus, 3,3 -((4-hydroxyphenyl)azanediyl)di(propanoic)acid ( 4) with an excess of methanol in the presence of a catalytic amount of sulfuric acid.
Subsequently, dihydrazide 6 was obtained through hydrazinolysis of dimethyl ester 5 in propan-2-ol under reflux (Scheme 1).Comparing the 1 H NMR spectrum of dihydrazide 6 with the 1 H NMR spectrum of diester 5, the characteristic signal of the ester groups was not observed at 3.54 ppm.A broad singlet integrated for four protons (4.17 ppm) and two singlets (8.61 and 8.98 ppm), each integrated for one proton, has been attributed to the NH2 and NH groups of two hydrazide fragments in the 1 H NMR spectrum of compound 6.
Hydrazones 7-13 were synthesized in good yields (58-94%) through the reaction of N-(4-hydroxyphenyl)-β-alanine hydrazide (3) with aromatic aldehydes in methanol at reflux temperature (Scheme 2).Hydrazones 14-17 were also obtained analogously, only after using heterocyclic aldehydes instead of aromatic aldehydes.The structures of hydrazones 7- 16 have been established mainly on the basis of 1 H and 13 C NMR spectra (Figures S1-S65).Theoretically, such compounds possessing amide and azomethine groups can exist as inseparable mixtures of four isomers.The amide group determined a splitting of resonances in the 1 H and 13 C NMR spectra due to the restricted rotation around the amide bond.The data of the 1 H and 13 C NMR spectra led us to conclude that geometrical isomers of the azomethine group were not observed, and in the DMSO-d6 solutions, hydrazones 7-16 existed as mixtures of E/Z isomers, where the Z isomer predominated due to the hindered rotation around the CO-NH bond.Subsequently, dihydrazide 6 was obtained through hydrazinolysis of dimethyl ester 5 in propan-2-ol under reflux (Scheme 1).Comparing the 1 H NMR spectrum of dihydrazide 6 with the 1 H NMR spectrum of diester 5, the characteristic signal of the ester groups was not observed at 3.54 ppm.A broad singlet integrated for four protons (4.17 ppm) and two singlets (8.61 and 8.98 ppm), each integrated for one proton, has been attributed to the NH 2 and NH groups of two hydrazide fragments in the 1 H NMR spectrum of compound 6.
Hydrazones 7-13 were synthesized in good yields (58-94%) through the reaction of N-(4-hydroxyphenyl)-β-alanine hydrazide (3) with aromatic aldehydes in methanol at reflux temperature (Scheme 2).Hydrazones 14-17 were also obtained analogously, only after using heterocyclic aldehydes instead of aromatic aldehydes.The structures of hydrazones 7- 16 have been established mainly on the basis of 1 H and 13 C NMR spectra (Figures S1-S65).Theoretically, such compounds possessing amide and azomethine groups can exist as inseparable mixtures of four isomers.The amide group determined a splitting of resonances in the 1 H and 13 C NMR spectra due to the restricted rotation around the amide bond.The data of the 1 H and 13 C NMR spectra led us to conclude that geometrical isomers of the azomethine group were not observed, and in the DMSO-d 6 solutions, hydrazones 7-16 existed as mixtures of E/Z isomers, where the Z isomer predominated due to the hindered rotation around the CO-NH bond.Subsequently, dihydrazide 6 was obtained through hydrazinolysis of dimethyl ester 5 in propan-2-ol under reflux (Scheme 1).Comparing the 1 H NMR spectrum of dihydrazide 6 with the 1 H NMR spectrum of diester 5, the characteristic signal of the ester groups was not observed at 3.54 ppm.A broad singlet integrated for four protons (4.17 ppm) and two singlets (8.61 and 8.98 ppm), each integrated for one proton, has been attributed to the NH2 and NH groups of two hydrazide fragments in the 1 H NMR spectrum of compound 6.
Hydrazones 7-13 were synthesized in good yields (58-94%) through the reaction of N-(4-hydroxyphenyl)-β-alanine hydrazide (3) with aromatic aldehydes in methanol at reflux temperature (Scheme 2).Hydrazones 14-17 were also obtained analogously, only after using heterocyclic aldehydes instead of aromatic aldehydes.The structures of hydrazones 7-16 have been established mainly on the basis of 1 H and 13 C NMR spectra (Figures S1-S65).Theoretically, such compounds possessing amide and azomethine groups can exist as inseparable mixtures of four isomers.The amide group determined a splitting of resonances in the 1 H and 13 C NMR spectra due to the restricted rotation around the amide bond.The data of the 1 H and 13 C NMR spectra led us to conclude that geometrical isomers of the azomethine group were not observed, and in the DMSO-d6 solutions, hydrazones 7-16 existed as mixtures of E/Z isomers, where the Z isomer predominated due to the hindered rotation around the CO-NH bond.sponding carbohydrazide 3, 2,5-hexanedione, 2-propanol, and a catalytic amount of glacial acetic acid (Scheme 2), whereas with isatin, hydrazide 3 formed a hydrazone-type compound 3-((4-hydroxyphenyl)amino)-N′-(2-oxoindolin-3-ylidene)propanehydrazide (18).
In the next stage of this work, condensation reactions of dihydrazide 6 with various carbonyl compounds were performed, during which a series of hydrazones 19 and 21-35, as well as oxadiazole, dimethylpyrazole, and dimethylpyrrole heterocyclic compounds 20, 36, and 37, were synthesized.Condensation of dihydrazide 6 with aromatic aldehydes and ketones gave the corresponding hydrazones 21-35 (Scheme 3) among the compounds due to the mode of substitution in the azomethine fragment.The presence of particular substitution patterns in the benzene ring as well as a mono substituent of the azomethine fragment caused the formation of geometrical isomers.Taking into account the two isomerism centers existing in each side chain, 10 isomers of compounds 21-35 can be formed.NMR did not provide conclusive information about the separate conformations but gave a time-averaged spectral view of the structures present in the solution.The restricted rotation around the CONH led to the formation in an isomeric mixture of hydrazones, where the Z isomer predominates.The obtained hydrazones 7-16 and 21-35 show double sets of resonances for the N=CH and CONH fragment protons with an intensity ratio of 0.35:0.65 ( 1 H NMR). The structures of similar compounds are described in our previous works [23][24][25][26].The synthesis of the compounds and their spectroscopic data are presented in the Supplementary Materials.

Antimicrobial activity of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives 2-37 against multidrug-resistant pathogens
Following the synthesis and characterization of novel 3-((4-hydroxyphenyl)amino)pro panoic acid derivatives 2-37, our primary objective was to investigate their antimicrobial activity against a variety of pathogenic bacterial and fungal organisms.The synthesized compounds were screened against diverse libraries of clinically relevant bacterial and fungal pathogens, harboring various multidrug-resistance phenotypes.The selection of isolates was undertaken to represent pathogens identified by the World Health Organization as high-priority ESKAPE group organisms, given their escalating levels of antimicrobial resistance in the clinical field.
Furthermore, the introduction of a 4-Cl substituent in the phenyl ring (compound 9) augmented activity directed toward Gram-positive bacteria while concurrently diminishing activity against Gram-negative pathogens, with the exception of A. baumannii.Notably, the introduction of a dimethylamino substituent in the phenyl ring (compound 10) resulted in a complete loss of antimicrobial activity against all tested bacterial strains.Moreover, the introduction of a 4-OH group (compound 11) or a 3,4,5-(CH 3 O) moiety (compound 12) failed to restore antimicrobial activity.Conversely, the introduction of a 1-naphthyl substituent (compound 13) resulted in the restoration of antimicrobial activity against S. aureus TCH-1516 and E. faecalis AR-0671 (MIC of 16 µg/mL), as well as E. coli AR-0001 (MIC of 32 µg/mL), K. pneumoniae AR-0003 (MIC of 64 µg/mL), and A. baumannii AR-0273 (MIC of 64 µg/mL).
Hydrazones 14-17, bearing various heterocyclic substituents, exhibited notably enhanced antimicrobial efficacy against bacterial and fungal pathogens.Specifically, compound 14, bearing a 2-thiophene substituent, demonstrated a broad spectrum of antibacterial and antifungal activity against all tested bacterial strains (MIC of 8-64 µg/mL).Notably, hydrazone 14 exhibited substantial activity against S. aureus TCH-1516 and E. faecalis AR-0671 (8 µg/mL), with comparatively weaker activity observed against E. coli AR-0001, K. pneumoniae AR-0003, P. aeruginosa AR-1114, and A. baumannii AR-0273 (64 µg/mL).The introduction of a nitro group into the thiophene backbone remarkably enhanced the antimicrobial activity of compound 15.This derivative displayed potent activity against S. aureus (MIC of 1 µg/mL) and E. faecalis AR-0671 (MIC < 0.5 µg/mL), as well as E. coli AR-0001 (MIC of 8 µg/mL), A. baumannii AR-0276 (MIC 16 µg/mL), and K. pneumoniae AR-003 (MIC of 32 µg/mL).Interestingly, the incorporation of the nitro group resulted in a reduction in the antifungal spectrum of compound 15, confining its activity solely to C. albicans.These findings underscore the structure-activity relationship of the hydrazone derivatives and highlight the potential of compound 15 as a potent antimicrobial agent with selectivity toward specific bacterial and fungal strains.The replacement of nitrothiophene substituent with nitrofurane (compound 16) preserved Gram-positive bacteria-targeted activity, while decreasing the antimicrobial spectrum against Gram-negative pathogens.On the other hand, the introduction of nitrofurane substitution enhanced the antifungal activity against C. albicans (MIC of 8 µg/mL), C. parapsilosis (MIC of 16 µg/mL), and C. auris strains (MIC of 32 µg/mL).Finally, dimethylpyrole containing derivative 17 showed no antibacterial activity (>64 µg/mL) against the majority of strains, although favorable activity was observed against A. baumannii (MIC of 16 µg/mL).Furthermore, strong antifungal activity was observed against drug-resistant Candida species (MIC of 8-16 µg/mL).Compounds with an indolinone moiety (compounds 18 and 19) had fully diminished antifungal and antibacterial activity.
Upon elucidating the structure-activity relationships of hydrazide derivatives, our investigation progressed toward the examination of symmetric dihydrazide-based transformations, postulating potential enhancements in antimicrobial properties.Specifically, dihydrazide 6 served as the starting point, undergoing a series of chemical transformations wherein it was condensed with diverse carbonyl compounds, yielding hydrazone, oxadiazole, dimethylpyrazole, and dimethylpyrrole heterocyclic derivatives, each harboring distinct structural substitutions.Of note, oxadiazole derivative 20 showed promising antimicrobial activity against S. aureus TCH-1516 and E. faecalis AR-0671, with a minimum inhibitory concentration (MIC) of 32 µg/mL.These compounds exhibited a lack of antimicrobial activity against Gram-negative pathogens and fungal strains.
We then evaluated whether the incorporation of heterocyclic substitutions would have an impact on the antimicrobial activity of synthesized compounds.Compound 21, bearing a 2-furyl ring, displayed enhanced antimicrobial efficacy against tested S. aureus and E. faecalis strains, with an MIC of 16 µg/mL.Additionally, compound 21 demonstrated activity against E. coli and K. pneumoniae, while showing no activity on P. aeruginosa (MIC >64 µg/mL).The replacement of furane with thiophene in compound 22 expanded the antimicrobial spectrum, particularly increasing the activity against tested P. aeruginosa, with an MIC of 64 µg/mL.Intriguingly, the introduction of a nitro group into the thiophene substituent, as observed in compound 23, resulted in a complete loss of antimicrobial activity against all tested bacterial and fungal strains.In contrast, the incorporation of a nitro substituent into furane, as observed in compound 24, remarkably restored antimicrobial activity against both Gram-positive and Gram-negative pathogens.Finally, the addition of a thien-3-yl moiety in compound 25 resulted in modest activity against Grampositive pathogens (MIC of 32 and 64 µg/mL) and negligible efficacy against Gram-negative pathogens or fungal strains.
Following the characterization of the impact of heterocycle substitution on the antimicrobial activity of dihydrazide derivatives, our investigation extended to the examination of how the inclusion of methyl or ethyl substitutions influences biological activity.Compound 26, bearing a CH 3 substituent, and compound 27, containing a C 2 H 5 substituent, demonstrated no discernible antimicrobial activity against all tested strains (MIC >64 µg/mL).Conversely, the incorporation of a phenyl substituent in compound 28 resulted in weak antimicrobial activity against E. coli (MIC of 32 µg/mL), K. pneumoniae (MIC of 64 µg/mL), and A. baumannii (MIC of 64 µg/mL).
Further exploration of the influence of various aromatic substituents on the antimicrobial activity of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives revealed that the inclusion of a phenyl substituent in compound 29 exhibited antimicrobial activity against S. aureus (MIC of 16 µg/mL) but not E. faecalis (MIC >64 µg/mL).The incorporation of a 4-NO 2 substitution in the phenyl ring in compound 30 led to enhanced activity against S. aureus and E. faecalis (MIC of 16 µg/mL), as well as E. coli (MIC of 32 µg/mL) and K. pneumoniae (MIC of 64 µg/mL).The substitution of a nitro group with 4-Cl resulted in compound 31 demonstrating increased activity against Gram-negative pathogens (MIC of 32-64 µg/mL), including efficacy against P. aeruginosa (MIC of 64 µg/mL) and A. baumannii (MIC of 32 µg/mL).
Notably, the incorporation of a basic substitution such as dimethylamino (compound 32) nullified activity against all tested organisms, except for A. baumannii (MIC of 16 µg/mL).Additionally, the introduction of a 4-OH substituent in compound 33 yielded substantial antimicrobial activity against both Gram-positive (MIC of 8-16 µg/mL) and Gram-negative pathogens (MIC of  µg/mL), including P. aeruginosa (MIC of 16 µg/mL) and A. baumannii (MIC of 16 µg/mL).The dimethylpyrazole derivative 36 demonstrated activity primarily against S. aureus and E. faecalis (MIC of 32 and 64 µg/mL, respectively), while the inclusion of dimethylpyrrole in compound 37 resulted in slightly expanded activity against E. coli (MIC of 16 µg/mL).
Collectively, these results demonstrate that 3-((4-hydroxyphenyl)amino)propanoic acid derivatives exhibit a structure-dependent and potent antimicrobial activity against ESKAPE group pathogens and drug-resistant fungi.

3-((4-hydroxyphenyl)amino)propanoic acid scaffold demonstrates favorable pharmacological ADME properties
After characterizing the antimicrobial activity of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives and establishing SAR relations, we further selected the most promising antibacterial and antifungal compounds and subjected them to in silico absorption, distribution, metabolism, and excretion (ADME) characterization to better understand the pharmacological properties of the 3-((4-hydroxyphenyl)amino)propanoic acid scaffold.
The compounds selected for in silico ADME prediction were chosen to represent a broad-spectrum antibacterial activity against multiple MDR strains.Among such compounds, 9, 13-16, 20-22, 24, 29, 30, 31, 33, and 36-37 were selected for in silico ADME analysis and compared to the reference antimicrobial drug, cefazolin (CEF) (Figure 1).For compounds with antifungal activity, 3-((4-hydroxyphenyl)amino)propanoic acid derivatives 14, 16, and 17 were selected and compared with fluconazole (FLU) (Figure 2).3).The fraction of sp 3 -hybridized carbon atoms (Csp 3 ) ranged from 0.1 to 0.43, reflecting differences in molecular rigidity.Compounds 14 and 15 showed a higher fraction of sp 3 hybridization, indicative of increased aliphatic character.The number of rotatable bonds ranged from 7 to 15, with compound 14 having the lowest and  3).The fraction of sp 3 -hybridized carbon atoms (Csp 3 ) ranged from 0.1 to 0.43, reflecting differences in molecular rigidity.Compounds 14 and 15 showed a higher fraction of sp 3 hybridization, indicative of increased aliphatic character.The number of rotatable bonds ranged from 7 to 15, with compound 14 having the lowest and compounds 30 and 31 having the highest numbers.Hydrogen bond acceptors and donors varied across compounds, with compound 24 having the highest number of acceptors (11) and compound 37 having the highest number of donors (3) (Table 3).Among the analyzed antibacterial compounds, eight (9, 13, 14, 29, 31, 36, and 37) were predicted to possess drug-like properties.Specifically, compounds 9 and 13 were predicted to exhibit the ability to passively permeate the blood-brain barrier (BBB) (Figure 1).These compounds displayed favorable physicochemical properties, including a molecular mass ranging from 317 to 333 g/mol, a suitable number of aromatic and heavy atoms, as well as an optimal count of rotatable bonds, and hydrogen acceptors and donors (Table 3).
Compounds 14, 29, 31, 36, and 37 demonstrated favorable gastrointestinal absorption (GI absorption) parameters (Figure 1, Table 4).Interestingly, only compound 37 was predicted to be a substrate of P-gp glycoprotein and to be actively exported from the CNS.
The in silico ADME prediction results for selected antibacterial compounds and their interaction with the human cytochrome P450 (CYP) system reveal distinct profiles across various CYP isoforms.Compounds 9, 13, 14, 15, 21, 22, 24, 29, 30, 31, 36, and 37 were investigated for their inhibitory effects on CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4.Compounds 9 and 13 exhibited significant inhibitory activity across all tested CYP isoforms, suggesting potential implications for drug metabolism and interactions.Compounds 14, 15, 21, 22, 24, 29, 30, 31, 36, and 37 displayed varying degrees of inhibition, highlighting the importance of considering multiple CYP isoforms in drug development.Compound 20 selectively inhibited CYP3A4, indicating specificity for this isoform.Compound 16 and the reference antibiotic CEF showed negligible inhibitory effects on the tested CYP isoforms.These findings contribute valuable insights into the potential pharmacokinetic interactions and metabolic fate of the investigated antibacterial compounds, aiding in the rational design of safer and more effective therapeutic agents.The molecular weight of the compounds ranges from 273.33 to 318.28 g/mol.Notably, compounds 14, 16, and fluconazole (FLU) exhibit varying heavy atom counts, ranging from 20 to 23.Aromatic heavy atom numbers range from 11 to 16, with FLU possessing the highest count.The fraction of sp 3 -hybridized carbon atoms (Csp3) varies among the compounds, indicating differences in molecular rigidity.Compounds 14, 16, and FLU display a fraction of 0.14, suggesting a comparable level of aliphatic character.The number of rotatable bonds ranges from 5 to 8, with compound 17 having the lowest and compound 16 having the highest (Table 6).This comprehensive molecular modeling provides valuable information for understanding the structural diversity and potential pharmacokinetic properties of the investigated antibacterial and antifungal 3-((4-hydroxyphenyl)amino)propanoic acid-based compounds.

Discussion
The rising prevalence of antimicrobial resistance among clinically significant bacterial and fungal pathogens underscores the need for urgent exploration of novel candidates and pharmacophores for subsequent pre-clinical evaluation.This study delineates a synthetic methodology designed to generate a diverse array of xenogeneic amino acid derivatives based on 3-((4-hydroxyphenyl)amino)propanoic acid, incorporating various aromatic and heterocyclic substitutions.Through antimicrobial activity characterization using genetically defined ESKAPE group pathogens harboring emerging resistance mechanisms, we demonstrated that the 3-((4-hydroxyphenyl)amino)propanoic acid-based pharmacophore exhibits promising activity against bacterial and fungal pathogens.
Various proteogenic and non-proteogenic amino acids play a crucial role in signaling, homeostasis, and protein synthesis in both eukaryotic and prokaryotic organisms, rendering the amino acid axis an attractive pathway for antimicrobial strategies [27][28][29].The utilization of amino acid derivatives as antimicrobials has been previously suggested by several researchers.Synthetic pathways and compounds have also been explored through diverse chemical approaches and model systems [30][31][32].Lopez et al. conducted a comprehensive investigation into the synthesis and antimicrobial activity of naphthoquinone moieties containing amino acid derivatives [30].Their study revealed promising antimicrobial efficacy against wild-type bacterial strains.Meanwhile, the research by Chui et al. delved into the antimicrobial properties of hydroxypropyltrimethyl ammonium chitosan derivatives bearing amino acid Schiff bases.Notably, this study successfully demonstrated robust antimicrobial activity, primarily targeting S. aureus and fungal strains [33].Despite the previously reported antimicrobial activity of various amino acid-based derivatives, it is noteworthy that a substantial portion of these studies predominantly involve wild-type and pan-susceptible bacterial isolates.This approach often overlooks the incorporation of multidrug-resistance mechanisms, thereby providing a less representative portrayal of the current resistance phenotypes circulating in clinical settings.Furthermore, drug-resistant bacterial and fungal pathogens frequently exhibit a spectrum of resistance mechanisms, capable of directly or indirectly modifying diverse chemical compounds or actively exporting them through the overexpression of efflux pumps.
In this study, we explored 3-((4-hydroxyphenyl)amino)propanoic acid derivatives for their antimicrobial efficacy against the prevalent ESKAPE pathogens (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) [34,35].Furthermore, by performing in silico ADME characterization using selected antibacterial and antifungal derivatives, we successfully demonstrated the pharmacological applicability of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives as promising drug-like hits suitable for future hit-to-lead optimization.The 4hydroxyphenyl substituent serves as a well-established pharmacophore frequently encountered in numerous FDA-approved pharmaceuticals.This moiety plays a pivotal role in augmenting the hydrophilicity of compounds, thereby potentially improving their solubility.Additionally, the 4-hydroxyphenyl radical contributes to enhanced reactivity by serving as a site for hydrogen bond formation.Given these characteristics, we hypothesized that substituted amino acid derivatives based on 4-hydroxyphenyl and propionic acid, incorporating diverse substituents, could yield potent antimicrobial activity against a spectrum of microbial pathogens.
We demonstrated that hydrazones 7-13 exhibited selective antimicrobial activity against methicillin-resistant S. aureus and vancomycin-resistant E. faecalis strains.Interestingly, no activity was observed against MDR Gram-negative bacterial isolates or drug-resistant strains, suggesting the Gram-positive bacteria-directed activity of hydrazones 7-13.Interestingly, the incorporation of heterocyclic substituents in hydrazonebased 3-((4-hydroxyphenyl)amino)propanoic acid derivatives ( [14][15][16][17] greatly enhanced the antimicrobial activity.These compounds showed activity against both Gram-positive and Gram-negative pathogens, as well as challenging fungal pathogens, including azoleresistant Candida auris.Among these compounds, hydrazone derivatives bearing nitro thiophene (15) and nitro furane (16) showed the most promising antibacterial activity, while compounds 14 and 17 bearing thiophene and dimethylpyrrole demonstrated promising antifungal activity against all tested isolates.Interestingly, the generation of dihydrazide derivatives with the identical substitutions decreased the antimicrobial activity.

Chemical synthesis General procedures
Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification.The reaction course and purity of the synthesized compounds were monitored by TLC using aluminum plates pre-coated with Silica gel with F 254 nm (Merck KGaA, Darmstadt, Germany).Melting points were determined with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and were uncorrected.IR spectra (ν, cm −1 ) were recorded on a Perkin-Elmer Spectrum BX FT-IR spectrometer (Perkin-Elmer Inc., Waltham, MA, USA) using KBr pellets.NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland).Chemical shifts were reported in (δ) ppm relative to tetramethylsilane (TMS), with the residual solvent as internal reference ([D 6 ]DMSO, δ = 2.50 ppm for 1 H and δ = 39.5 ppm for 13 C).Data are reported as follows: chemical shift, multiplicity, coupling constant [Hz], integration, and assignment.Elemental analyses (C, H, and N) were conducted using the Elemental Analyzer CE-440, and their results were found to be in good agreement (±0.3%) with the calculated values.Mass spectra were measured on a Bruker maXis 4G mass spectrometer.

General procedure for the preparation of hydrazones 7-13
To a solution of hydrazide 3 (1.5 mmol) in methanol (11 mL), the corresponding aromatic aldehyde was added (1.65 mmol); the mixture was heated at reflux temperature for 2 h, then cooled down, and the formed precipitate was filtered off, washed with methanol and diethyl ether, and recrystallized from 1,4-dioxane.
Dark green powder, yield 0.31 g (64%), m.p.To a solution of dihydrazide 6 (0.3 g, 1.1 mmol) in 2-propanol (17 mL), hexane-2,5dione (0.50 g, 4.4 mmol) and a catalytic amount of acetic acid (0.05 mL) were added, and the mixture was heated under reflux for 5 h, then cooled down, and diluted with water (20 mL); the formed precipitate was filtered off, washed with water, and recrystallized from a mixture of 2-propanol

Microbial strains and culture conditions
The multidrug-resistant S. aureus strain TCH 1516 [USA 300-HOU-MR] was obtained from the American Type Culture Collection (ATCC).Vancomycin-resistant E. faecalis and drug-resistant Candida species strains were acquired from the Centers for Disease Control and Prevention (CDC) AR isolate bank.Prior to the initiation of this study, all microbial strains were stored in commercial cryopreservation systems at a temperature of −80 • C. The strains were cultivated on Columbia sheep blood agar (Becton Dickinson, Franklin Lakes, NJ, USA), or potato dextrose agar (PDA) for Candida (Becton Dickinson, USA).

Minimal inhibitory concentration determination
The antimicrobial activity of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives was assessed using the broth microdilution method, following the guidelines outlined by the Clinical Laboratory Standards Institute (CLSI), with modifications [18].In brief, the compounds were dissolved in dimethylsulfoxide (DMSO) to attain a final concentration of 25-30 mg/mL.Dilution series were prepared in deep 96-well microplates to achieve a two-fold concentration range of 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 µg/mL, utilizing cation-adjusted Mueller-Hinton broth (CAMHB) as the growth medium.For Candida screening, dilutions were performed in RMPI/MOPS media.The microplates containing the dilution series were then inoculated with fresh cultures of each tested organism to reach a final concentration of 5 × 10 4 CFU (colony-forming units) of the test organism in media containing 1% DMSO and 1× drug concentration, with a volume of 200 µL per well.Wells that were inoculated with media containing 1% DMSO served as positive controls.Subsequently, the microplates were incubated at 35 ± 1 • C for 18 ± 2 h.Following the incubation period, the plates were examined using a manual microplate viewer (Sensititre Manual Viewbox, United States).The minimal inhibitory concentration (MIC) was defined as the lowest concentration (µg/mL) of the tested drug that completely inhibited the growth of the test organism.All experiments were conducted in duplicate with three technical replicates for each condition.

In silico ADME prediction
The structures of selected compounds were converted to SMILE structures and used for in silico ADME prediction using SwissAdme online tool (http://www.swissadme.ch/faq.php, accessed on 1 January 2024).

Conclusions
This study demonstrates that novel substituted amino acid derivatives, derived from 3-((4-hydroxyphenyl)amino)propanoic acid, exhibit promising antimicrobial activity against ESKAPE group pathogens and drug-resistant fungal strains, including C. auris.The investigation underscores that amino acid derivatives bearing a 4-hydroxyphenyl core, along with heterocyclic substituents, show potent broad-spectrum antibacterial and antifungal activity and drug-like ADME properties.This study provides foundational and synthetic procedures for subsequent compound generation, paving the way for hit-to-lead optimization and early and structure-activity relation data.Further investigations are crucial to enhance our understanding of the in vitro and in vivo safety, biological availability, and tolerability of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives, as well as additional compounds based on this pharmacophore.

Figure 1 .
Figure 1.The boiled egg model illustrates the ADME properties of selected antibacterial derivatives of 3-((4-hydroxyphenyl)amino)propanoic acid.Blue dots indicate compounds predicted to be P-gp substrates, potentially undergoing active efflux from the central nervous system (CNS).Conversely, red dots indicate compounds predicted not to be P-gp substrates and not expected to undergo active efflux from the CNS.

Figure 1 .
Figure 1.The boiled egg model illustrates the ADME properties of selected antibacterial derivatives of 3-((4-hydroxyphenyl)amino)propanoic acid.Blue dots indicate compounds predicted to be P-gp substrates, potentially undergoing active efflux from the central nervous system (CNS).Conversely, red dots indicate compounds predicted not to be P-gp substrates and not expected to undergo active efflux from the CNS.Antibiotics 2024, 13, x FOR PEER REVIEW 10 of 25

Figure 2 .
Figure 2. The boiled egg model illustrates the ADME properties of selected antifungal derivatives of 3-((4-hydroxyphenyl)amino)propanoic acid.Blue dots indicate compounds predicted to be P--gp substrates, potentially undergoing active efflux from the central nervous system (CNS).Conversely, red dots indicate compounds predicted not to be P-gp substrates and not expected to undergo active efflux from the CNS.The physicochemical properties of selected antibacterial 3-((4-hydroxyphenyl)amino)propanoic acid derivatives were assessed to gain insights into their molecular characteristics.The molecular weight (MW) of the compounds ranged from 289.35 to 547.52 g/mol, with compound 30 exhibiting the highest MW.The number of heavy atoms varied between 20 and 40, with compound 14 having the lowest and compound 30 having the highest number (Table3).The fraction of sp3 -hybridized carbon atoms (Csp 3 ) ranged from 0.1 to 0.43, reflecting differences in molecular rigidity.Compounds 14 and 15 showed a higher fraction of sp 3 hybridization, indicative of increased aliphatic character.The number of rotatable bonds ranged from 7 to 15, with compound 14 having the lowest and

Figure 2 .
Figure 2. The boiled egg model illustrates the ADME properties of selected antifungal derivatives of 3-((4-hydroxyphenyl)amino)propanoic acid.Blue dots indicate compounds predicted to be P--gp substrates, potentially undergoing active efflux from the central nervous system (CNS).Conversely, red dots indicate compounds predicted not to be P-gp substrates and not expected to undergo active efflux from the CNS.The physicochemical properties of selected antibacterial 3-((4-hydroxyphenyl)amino)p ropanoic acid derivatives were assessed to gain insights into their molecular characteristics.The molecular weight (MW) of the compounds ranged from 289.35 to 547.52 g/mol, with compound 30 exhibiting the highest MW.The number of heavy atoms varied between 20 and 40, with compound 14 having the lowest and compound 30 having the highest